Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli BL21 with a 3C protease-removable N-terminal hexahistidine tag | Teredinibacter turnerae |
gene TERTU_4506, recombinant expression of codon-optimized N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3) | Teredinibacter turnerae |
Crystallization (Comment) | Organism |
---|---|
purified wild-type enzyme TtGH8 uncomplexed and complexed with xylobiose and xylotriose, and mutant D281N complexed with xylohexaose, enzyme TtGH8 is mixed with well solution containing 0.1 M sodium acetate pH 4.6-5.2, 0.2 M NaCl, 14-24% PEG 6000, method optimization screening, X-ray diffraction structure determination and analysis at 1.4-1.8 A resolution, molecular replacement using Bacillus halodurans C-125 structure (PDB ID 1wu4) as the search model | Teredinibacter turnerae |
the three-dimensional structure of the enzyme is solved in uncomplexed and xylobiose-, xylotriose- and xylohexaose-bound forms at approximately 1.5 A resolution | Teredinibacter turnerae |
Protein Variants | Comment | Organism |
---|---|---|
D281N | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme. The TtGH8 D281N-beta-1,4-xylohexaose complex structure reveals that, the -1 subsite sugar is in a completely ring-flipped, southern hemisphere 1C4 chair conformation. Although this allows access to a hexasaccharide complex structure, the ring-flipped -1 sugar is unlikely to be representative of a catalytically relevant conformation since its position neither allows protonation of the leaving group by Glu73 nor is there a potential reactive water. In the 1C4 chair conformation the now axial (and down) O2 occupies the position that should instead be occupied by the nucleophilic water | Teredinibacter turnerae |
additional information | the catalytic domain is identified and truncated to remove the original signal peptide and linker region and to include an N-terminal hexahistidine tag and 3C protease cleavage site | Teredinibacter turnerae |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
marine beta-1,3-beta-1,4-xylan + H2O | Teredinibacter turnerae | - |
? | - |
? | |
additional information | Teredinibacter turnerae | enzyme TtGH8 is effective at the degradation of xylan-based substrates, notably beta-1,4-xylan and mixed-linkage (beta-1,3/beta-1,4) marine xylan | ? | - |
? | |
additional information | Teredinibacter turnerae ATCC 39867 | enzyme TtGH8 is effective at the degradation of xylan-based substrates, notably beta-1,4-xylan and mixed-linkage (beta-1,3/beta-1,4) marine xylan | ? | - |
? | |
additional information | Teredinibacter turnerae T7901 | enzyme TtGH8 is effective at the degradation of xylan-based substrates, notably beta-1,4-xylan and mixed-linkage (beta-1,3/beta-1,4) marine xylan | ? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Teredinibacter turnerae | C5BJ89 | - |
- |
Teredinibacter turnerae ATCC 39867 | C5BJ89 | - |
- |
Teredinibacter turnerae T7901 | C5BJ89 | - |
- |
Purification (Comment) | Organism |
---|---|
- |
Teredinibacter turnerae |
recombinant N-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage by 3C protease, another step of nickel affinity chromatography, and gel filtration | Teredinibacter turnerae |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
arabinoxylan + H2O | - |
Teredinibacter turnerae | ? | - |
? | |
arabinoxylan + H2O | - |
Teredinibacter turnerae ATCC 39867 | ? | - |
? | |
arabinoxylan + H2O | - |
Teredinibacter turnerae T7901 | ? | - |
? | |
beta-1,4-xylan + H2O | - |
Teredinibacter turnerae | ? | - |
? | |
beta-1,4-xylan + H2O | - |
Teredinibacter turnerae ATCC 39867 | ? | - |
? | |
beta-1,4-xylan + H2O | - |
Teredinibacter turnerae T7901 | ? | - |
? | |
beta-1,4-xylohexaose + H2O | - |
Teredinibacter turnerae | ? | - |
? | |
beta-1,4-xylopentaose + H2O | - |
Teredinibacter turnerae | ? | - |
? | |
beta-1,4-xylotetraose + H2O | - |
Teredinibacter turnerae | ? | - |
? | |
birchwood xylan + H2O | - |
Teredinibacter turnerae | ? | - |
? | |
marine beta-1,3-beta-1,4-xylan + H2O | - |
Teredinibacter turnerae | ? | - |
? | |
mixed-linkage (beta-1,3,beta-1,4) xylan + H2O | maximal activity against mixed-linkage polymeric xylans | Teredinibacter turnerae | ? | - |
? | |
additional information | enzyme TtGH8 is effective at the degradation of xylan-based substrates, notably beta-1,4-xylan and mixed-linkage (beta-1,3/beta-1,4) marine xylan | Teredinibacter turnerae | ? | - |
? | |
additional information | the enzyme TtGH8 is a xylan-active endoxylanase with six catalytically relevant subsites and notably a maximal activity towards mixed-linkage (beta-1,3/beta-1,4) marine xylan, TtGH8 catalyses the hydrolysis of beta-1,4-xylohexaose and displays maximal activity against mixed-linked marine polymeric xylans | Teredinibacter turnerae | ? | - |
? | |
additional information | enzyme TtGH8 is effective at the degradation of xylan-based substrates, notably beta-1,4-xylan and mixed-linkage (beta-1,3/beta-1,4) marine xylan | Teredinibacter turnerae ATCC 39867 | ? | - |
? | |
additional information | the enzyme TtGH8 is a xylan-active endoxylanase with six catalytically relevant subsites and notably a maximal activity towards mixed-linkage (beta-1,3/beta-1,4) marine xylan, TtGH8 catalyses the hydrolysis of beta-1,4-xylohexaose and displays maximal activity against mixed-linked marine polymeric xylans | Teredinibacter turnerae ATCC 39867 | ? | - |
? | |
additional information | enzyme TtGH8 is effective at the degradation of xylan-based substrates, notably beta-1,4-xylan and mixed-linkage (beta-1,3/beta-1,4) marine xylan | Teredinibacter turnerae T7901 | ? | - |
? | |
additional information | the enzyme TtGH8 is a xylan-active endoxylanase with six catalytically relevant subsites and notably a maximal activity towards mixed-linkage (beta-1,3/beta-1,4) marine xylan, TtGH8 catalyses the hydrolysis of beta-1,4-xylohexaose and displays maximal activity against mixed-linked marine polymeric xylans | Teredinibacter turnerae T7901 | ? | - |
? | |
wheat arabinoxylan + H2O | - |
Teredinibacter turnerae | ? | - |
? | |
wheat arabinoxylan + H2O | - |
Teredinibacter turnerae ATCC 39867 | ? | - |
? | |
wheat arabinoxylan + H2O | - |
Teredinibacter turnerae T7901 | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | enzyme three-dimensional structure analysis | Teredinibacter turnerae |
Synonyms | Comment | Organism |
---|---|---|
endoxylanase | - |
Teredinibacter turnerae |
glycoside hydrolase family 8 domain protein | UniProt | Teredinibacter turnerae |
TERTU_4506 | - |
Teredinibacter turnerae |
TtGH8 | - |
Teredinibacter turnerae |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Teredinibacter turnerae |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
55.2 | - |
melting temperature of apoenzyme | Teredinibacter turnerae |
57.3 | - |
apoenzyme with xylan | Teredinibacter turnerae |
60.1 | - |
apoenzyme with xylohexaose | Teredinibacter turnerae |
90 | - |
purified recombinant enzyme, 20 min, inactivation | Teredinibacter turnerae |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6 | - |
assay at | Teredinibacter turnerae |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to the glycoside hydrolase family 8, GH8 | Teredinibacter turnerae |
additional information | the enzyme has six catalytically relevant subsites, structural analysis of substrate-binding sites and the distortions of xylose within the catalytic centre, overview. Enzymatic glycoside hydrolysis involves the distortion of the reactive, -1 subsite, sugar into a variety of skew-boat and boat conformations, reflecting the requirements of inline attack and the stereoelectronic requirements of an oxocarbenium-ion-like transition state | Teredinibacter turnerae |
physiological function | primary role in the degradation of marine polysaccharides | Teredinibacter turnerae |
physiological function | the enzyme plays a primary role in the degradation of marine polysaccharides, potential of Teredinibacter turnerae for effective and diverse biomass degradation | Teredinibacter turnerae |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.3 | - |
marine beta-1,3-beta-1,4-xylan | pH 6.0, 37°C, recombinant enzyme mutant D281N | Teredinibacter turnerae | |
10.17 | - |
beta-1,4-xylotetraose | pH 6.0, 37°C, recombinant wild-type enzyme | Teredinibacter turnerae | |
10.2 | - |
beta-1,4-xylotetraose | 37°C, pH 6.0 | Teredinibacter turnerae | |
105 | - |
arabinoxylan | pH 6.0, 37°C, recombinant wild-type enzyme | Teredinibacter turnerae | |
105 | - |
wheat arabinoxylan | 37°C, pH 6.0 | Teredinibacter turnerae | |
233 | - |
beta-1,4-xylopentaose | 37°C, pH 6.0 | Teredinibacter turnerae | |
233.33 | - |
beta-1,4-xylopentaose | pH 6.0, 37°C, recombinant wild-type enzyme | Teredinibacter turnerae | |
300 | - |
birchwood xylan | pH 6.0, 37°C, recombinant wild-type enzyme | Teredinibacter turnerae | |
300 | - |
birchwood xylan | 37°C, pH 6.0 | Teredinibacter turnerae | |
1250 | - |
beta-1,4-xylohexaose | 37°C, pH 6.0 | Teredinibacter turnerae | |
1259.26 | - |
beta-1,4-xylohexaose | pH 6.0, 37°C, recombinant wild-type enzyme | Teredinibacter turnerae | |
2666.67 | - |
marine beta-1,3-beta-1,4-xylan | pH 6.0, 37°C, recombinant wild-type enzyme | Teredinibacter turnerae | |
2666.7 | - |
mixed-linkage (beta-1,3,beta-1,4) xylan | 37°C, pH 6.0 | Teredinibacter turnerae |